CD44 Ligation Induces Caspase-Independent Cell Death Via a Novel Calpain/ AIF Pathwayin Human Erythroleukemiacells

Oncogene (2006) 25, 5741–5751 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE CD44 ligation induces caspase-independent cell death via a novel calpain/ AIF pathwayin human erythroleukemiacells C Artus1, E Maquarre1, RS Moubarak2, C Delettre2, C Jasmin1, SA Susin2 and J Robert-Le´ ze´ ne` s1 1INSERM U602, Hoˆpital Paul Brousse, Villejuif, France and 2Apoptose et Syste`me Immunitaire Group, Institut Pasteur, Paris, France Ligation of the cell surface molecule CD44 byanti-CD44 CD44 with specific anti-CD44 monoclonal antibodies monoclonal antibodies (mAbs) has been shown to induce (mAbs) can reverse differentiation blockage of the cell differentiation, cell growth inhibition and in some leukemia cells in primary blasts from patients with cases, apoptosis in myeloid leukemic cells. We report, AML-M1 to AML-M5 subtypes (Charrad et al., 1999). herein, that exposure of human erythroleukemic HEL More recently, the anti-CD44 mAb HI44a has been cells to the anti-CD44 mAb A3D8 resulted in cell growth reported to induce both differentiation and apoptosis in inhibition followed bycaspase-independent apoptosis-like AML cells (Song et al., 2004). In addition, the two cell death. This process was associated with the disruption activating anti-CD44 mAbs H90 and A3D8 trigger of mitochondrial membrane potential (DWm), the mito- terminal differentiation in several human myeloid chondrial release of apoptosis-inducing factor (AIF), but leukemia cell lines, strongly inhibit their proliferation not of cytochrome c, and the nuclear translocation of AIF. and, in some cases, induce apoptotic cell death (Charrad All these effects including cell death, loss of mitochondrial et al., 2002). Downregulation of c-Jun (Zada et al., 2003) DWm and AIF release were blocked bypretreatment with and c-Myc (Song et al., 2004) expression and upregula- the poly(ADP-ribose) polymerase inhibitor isoquinoline. tion of the cyclin-dependent kinase inhibitor p27kipÀ1 A significant protection against cell death was also (Gadhoum et al., 2004b) have recently been suggested to observed byusing small interfering RNA for AIF. explain the inhibition of cell proliferation upon CD44 Moreover, we show that calpain protease was activated ligation in AML cells. Recent data show that the anti- before the appearance of apoptosis, and that calpain CD44 mAb A3D8 induces apoptosis via caspase- and inhibitors or transfection of calpain-siRNA decrease serine-protease-dependent pathways in acute promyelo- A3D8-induced cell death, and block AIF release. These cytic leukemia NB4 cells (Maquarre et al., 2005). data suggest that CD44 ligation triggers a novel caspase- However, the signaling pathway by which anti-CD44 independent cell death pathwayvia calpain-dependent AIF mAbs induce cell death in other AML cells remains release in erythroleukemic HEL cells. undefined. Understanding the mechanism of CD44- Oncogene (2006) 25, 5741–5751. doi:10.1038/sj.onc.1209581; induced apoptosis would help in identifying novel published online 24 April 2006 therapeutic targets potentially useful in clinical trials. Cell death patterns have been divided into pro- Keywords: CD44; apoptosis; calpain; AIF; erythro- grammed cell death (PCD) or apoptosis and accidental leukemia or passive necrotic cell death (Leist and Jaattela, 2001). Apoptosis is actually the best characterized type of PCD: cells display membrane blebbing, loss of the asymmetry of phosphatidylserine (PS) in the plasma membrane, nuclear fragmentation, and activated Introduction caspases, a family of cell-suicide cysteine proteases (Nicholson, 1999). The biochemical activation of CD44 is a cell surface antigen that demonstrates cell apoptosis occurs via two major pathways: the intrinsic adhesion and signaling function (Ponta et al., 2003). pathway, initiated by the mitochondrial release of CD44 is involved in normal myelopoiesis and lympho- cytochrome c, resulting in the activation of caspase-9; poiesis and is also expressed on leukemia blasts in all and the extrinsic pathway, initiated by the cell surface acute myeloid leukemia (AML) subtypes (Ghaffari death receptors such as Fas that leads to the activation et al., 1999). It has been reported that the ligation of of caspase-8 or -10 (Sun et al., 1999). Both pathways converge and activate executioner caspase-3, which then cleaves intracellular protein substrates and causes Correspondence: Dr J Robert-Le´ ze´ ne` s, INSERM U602, Hoˆ pital Paul- cell death. There is now accumulating evidence indi- Brousse, 14 avenue Paul Vaillant Couturier, 94807 Villejuif cedex, cating that PCD can also occur in complete absence France. E-mail: [email protected] and independently of caspase activation (Johnson, Received 27 October 2005; revised 16 February 2006; accepted 8 March 2000; Jaattela and Tschopp, 2003; Lockshin and 2006; published online 24 April 2006 Zakeri, 2004; Broker et al., 2005). Several models of CD44-induced cell death in erythroleukemia cells CArtuset al 5742 caspase-independent PCD have been described. They Results include autophagy, paraptosis, mitotic catastrophe or the descriptive models of apoptosis-like and necrosis- Effect of CD44 ligation with A3D8 antibody in HEL cells like PCD (Broker et al., 2005). Caspase-independent By using A3D8 anti-CD44 mAb, preliminary results cell death pathways are important mechanisms for showed that HEL cells expressed CD44 as evaluated by triggering a response to cytotoxic agents or other death flow cytometry analysis (data not shown). When A3D8 stimuli when the caspase-mediated routes fail. These mAb was added to HEL cells for 4 days, a pronounced pathways may involve organelles such as lysosomes inhibition of cell proliferation was observed (Figure 1a, and the endoplasmic reticulum via the activation of upper panel). When two other anti-CD44 H90 and J173 non-caspases proteases such as cathepsins or calpains mAbs were tested, no such effects were observed (data (Johnson, 2000). However, the mitochondrion plays a not shown). The inhibitory effect of A3D8 was time- central role in caspase-independent cell death as in and-concentration-dependent. Thus, the decrease of cell caspase-dependent apoptosis (Bras et al., 2005). number appeared within 1–2 days and was most Key events in classic apoptosis as well as in other pronounced at high concentrations of A3D8 (3 mg/ml). forms of cell death are the disruption of mitochondrial At this latter concentration, a decrease in viable cells function and the release of apoptogenic proteins from occurred at day 3 concomitantly with appearance of cell the intermembrane space (IMS) of mitochondria death (Figure 1a, lower panel). Cell death was found to (Henry-Mowatt et al., 2004; Saelens et al., 2004). be also dose-dependent and reached 25–30% at 3 mg/ml Although the process of the mitochondrial outer of A3D8 by day 4. With further exposure, the membrane permeabilization (MOMP) is mainly con- proportion of dead cells increased up to about 60% by trolled by the members of the Bcl-2 family, the exact day 5 (data not shown). mechanisms responsible of this process remain contro- We next investigated whether A3D8-induced inhibi- versial (Donovan and Cotter, 2004). Whatever the tion of proliferation was owing to a cell cycle perturba- mechanism required for MOMP, release of mitochon- tion. A marked accumulation of HEL cells in S phase drial proteins causes most forms of caspase-dependent or -independent cell death. Mitochondrial proteins that induce caspase-dependent apoptosis include cytochrome c and two other proteins, Smac/Diablo and Omi/HtrA2, which antagonize the inhibitors of apoptosis (IAPs). Mitochondria can also release EndoG and apoptosis- inducing factor (AIF), one of the major mediators of cell death involved in caspase-independent apoptosis (Joza et al., 2001; Cande et al., 2004; Cregan et al., 2004). Apoptosis-inducing factor is expressed as a precursor of 67 kDa, which is addressed and compartmentalized into mitochondria by two-mitochondrial localization sequences located within the N-terminal prodomain of the protein (Susin et al., 1999). Once in mitochondria, the full-length AIF is processed and the prodomain removed, giving rise to a mature form of B57 kDa (Otera et al., 2005). Under physiological conditions, AIF is a mitochondrial FAD-dependent oxidoreductase that plays a role in oxidative phosphorylation (Miramar et al., 2001). However, after a cellular insult, AIF is cleaved by calpains and/or cathepsins (Polster et al., 2005; Yuste et al., 2005) and translocates from mitochondria to cytosol and to nucleus where it causes, in a caspase-independent fashion, chromatin condensa- tion and large-scale (B50 kb) DNA fragmentation (Susin et al., 1999). In the present study, we investigated the nature of the apoptotic signals mediated by CD44 ligation with the specific A3D8 mAb in human erythroleukemic HEL cells, a model of M6 subtype of AML that is usually refractory to conventional drugs. Our data reveal that Figure 1 Anti-CD44 mAb A3D8 induces cell growth inhibition CD44 ligation by A8D8 induced caspase-independent followed by cell death in HEL cells. (a) HEL cells were seeded at apoptosis-like cell death in HEL cells via AIF transloca- 2 Â 105 cells/ml and treated with various concentrations of A3D8 tion to nuclei and poly (ADP-ribose) polymerase (0.5–3 mg/ml). Control HEL cells were treated with IgG1. Trypan blue test was used to estimate cell growth (top) and cell death (PARP) activation. We also show that calpain activa- (bottom) along 4 days of treatment.

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